Cytokine-like proteins

ABSTRACT

A full-length cDNA corresponding to an EST (AA418955), which does not show any homology to other proteins in the database but has a weak homology to G-CSF, has been successfully isolated by synthesizing printers based on the EST sequence, and effecting PCR-cloning from a human fetal spleen library. Sequencing of the thus-isolated cDNA and analysis of its structure revealed that the cDNA has typical characteristics of a factor belonging to the IL-6/G-CSF/MGF family. It is also found out that the culture supernatant of said sequence-transfected CHO cells shows a proliferation supporting activity towards bone marrow cells in the coexistence of kit ligand.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of InternationalPatent Application No. PCT/JP99/01997, filed Apr. 14, 1999, and claimspriority from Japanese Patent Application No. 10/121805, filed Apr. 14,1998.

FIELD OF THE INVENTION

[0002] This invention relates to a novel cytokine-like protein and theencoding genie.

[0003] 1. Background of the Invention

[0004] Cytokines are multi-functional cell growth/differentiationinducing factors controlling immune and hematopoietic reactions. Theseries of factors composing cytokines, are mainly produced by activatedT cells, macrophages, or stromal cells and connect the cells of thelymphoid system and the hematopoietic system in a network, regulatingthe proliferation, differentiation, and functions of these cells. Sofar, a number of factors have been isolated as cytokines and apart fromthe factors themselves, antibodies and receptor molecules of thosefactors, or antibodies against those receptors, have been developed astherapeutic drugs and are in actual use.

[0005] For example, G-CSF, which has a neutrophil-proliferatingfunction, is already in use as a drug for many diseases and leukopeniaresulting from the treatment of these diseases (K. Welte et al., thefirst 10 years Blood Sep. 15 1996; 88:1907-1929 and also referGENDAIKAGAKU ZOUKAN no. 18, Cytokine, edited by TOSHIAKI OHSAWA, 1990,published by TOKYO KAGAKU DOUJIN). Furthermore, an antibody against thereceptor of IL-6, which acts in immune functions and inflammation, isbeing developed as a potential therapeutic drug against rheumatism andleukemia.

[0006] 2. Summary of the Invention

[0007] The present invention provides a novel cytokine-like protein andthe encoding DNA. It also provides a vector into which the DNA has beeninserted, a transformant carrying the DNA, and also a method forproducing a recombinant protein using the transformant. Furthermore,screening methods for a compound, which binds to the protein andregulates the activity, and the uses of the protein and the compoundsregulating its function as pharmaceutical drugs, are also provided bythe present invention.

[0008] Most cytokines known so far, have a conserved characteristic suchas a W S Motif (Idzerda, R L et al., J Exp Med 1990 Mar 1; 171 (3)861-873), and form a super-family of cytokine receptors. Although thecytokine itself, which is the ligand, does not have a conservedcharacteristic or homology compared to the receptor, some groups have anextremely weak homology, hinting of a close stereoscopic structure.EPO/TPO family and IL-6/G-CSF/MGF family can be taken as examples. Thepresent inventors, thinking that unknown, yet unidentified genes mayexist in these families, attempted to isolate an unknown cytokinebelonging to these families.

[0009] Specifically, it was found that the EST (AA418955) sequence,which does not show any homology to other proteins in the Database, hasa weak homology to G-SCF and constructed primers based on that sequence,and did PCR cloning from a human fetal spleen library. As a result, thepresent inventors succeeded in isolating a full-length cDNAcorresponding to the EST (this clone was named SGRF). Also, the isolatedSGRF cDNA was sequenced and the structure analyzed to find that theisolated cDNA has the typical characteristics of a factor belonging tothe IL-6/G-CSF/MGF family. Furthermore, the inventors analyzed theactivity of the SGRF protein to find that the culture supernatant ofSGRF-transfected CHO cells has a proliferation supporting activitytowards specific bone marrow cells in the presence of mouse kit ligand.The isolated SGRF protein itself may be applicable for the preventionand treatment of diseases of the lymphoid and hemiiatopoictic systemsand for diseases related to defective cell growth. Also it is possibleto use this protein for the screening of other factors related to thelymphoid and hematopoietic systems and as a drug candidate compound fordiseases of those systems.

[0010] Namely, this invention relates to a novel cytokine-like proteinSGRF and the encoding gene, their production as well as the use of theprotein in the screening of drugs and drug candidate compounds. Morespecifically,

[0011] 1. a protein comprising the amino acid sequence of SEQ ID NO:1,or said sequence in which one or more amino acids are replaced, deleted,added, and(/or inserted,

[0012] 2. a protein encoded by a DNA hybridizing with the DNA comprisingthe nucleotide sequence of SEQ ID NO:2, which is functionally equivalentto the protein having the amino acid sequence of SEQ ID NO:1,

[0013] 3. a DNA encoding the protein of (1) or (2),

[0014] 4. the DNA of (3), which contains the coding region of thenucleotide sequence of SEQ ID NO:2,

[0015] 5. a vector in which the DNA of (3) or (4) is inserted,

[0016] 6. a transformant carrying, in an expressible manner, the DNA of(3) or (4),

[0017] 7. a method for producing the protein of (1) or (2), whichcomprises the culturing of the transformant of (6),

[0018] 8. a method for screening a compound which can bind to theprotein of (1) or (2), the method comprising the steps of:

[0019] (a) exposing a test sample to the protein of (1) or (2) or itspartial peptide;

[0020] (b) detecting the binding activity between the test compound andsaid protein or its partial peptide; and

[0021] (c) selecting a compound having a binding activity to saidprotein,

[0022] 9. a compound which can bind to the protein of (1) or (2),

[0023] 10. the compound of (9) which is obtainable by the method of (8),

[0024] 11. a method for screening a compound which can promote orinhibit activity of the protein of (l) or (2), the method comprising thesteps of:

[0025] (a) exposing the protein of (1) or (2) and the kit ligand tomammalian bone marrow cells under the absence of a test compound;

[0026] (b) detecting the proliferation of said bone marrow cells; and

[0027] (c) selecting a compound which promotes or inhibits theproliferation of bone marrow cells in comparison with the assay underthe presence of the test sample,

[0028] 12. the method of (11), wherein the bone marrow cells are Linnegative, Sca-1 positive, c-kit positive, and CD34 positive,

[0029] 13. a compound which promotes or inhibits the activity of theprotein of (1) or (2),

[0030] 14. the compound of (13) which is obtainable by the method of(I 1) or (12),

[0031] 15. a pharmaceutical composition comprising the protein of (I) or(2) as an active component,

[0032] 16. a promoter or inhibitor of the protein of (1) or (2) whereinthe active component is the compound of (13) or (14),

[0033] 17. an antibody which can bind to the protein of (1) or (2), and

[0034] 18. a DNA comprising at least 15 nucleotides, which canspecifically hybridize with the DNA comprising the nucleotide sequenceof SEQ ID NO:2.

[0035] The present invention relates to a novel cytokine-like protein.The nucleotide sequence of the cDNA encoding the protein named “SGRF”,which is included in the protein of the present invention is shown inSEQ ID NO:2; the amino-acid sequence of said protein in SEQ ID NO:1.

[0036] So far, in mammals, IL-6 and G-CSF have been reported as factorsthought to belong to the IL-6/G-CSF/MGF family. The “SGRF” cDNA isolatedby the present inventors, had in its 3′ non-coding region, four mRNAdestabilizing sequences (Lagnando C A, Brown C Y, Goodall G J (1994)Mol. Cell. Biol. 14, 7984-7995) called ARE (AT Rich element), often seenin cytokine mRNAs. The consensus sequence preserved in theIL-6/G-CSF/MGF family was also roughly maintained (FIG. 3). From thesefacts, “SGRF” can be assumed to be a novel factor belonging to theIL-6/G-CSF/MGF family.

[0037] The “SGRF” expression in human normal tissue as detected bynorthern-blot analysis is extremely localized, and was seen in thetestis, lymph nodes, and thymus, being not present in a detectable levelin other tissues (FIG. 4). Even in tissues where expression wasdetected, the expression level was assumed to be very low. An EST(U38443), a partial fraction of “SGRF”, which is normally hardlyexpressed, is reportedly induced following activation in a T cell-line(Jurkat) (Yatindra Prashar, Sherman M. Weissman (1996) Proc. Natl. Acad.Sci. USA 93:659-663). From this fact and from the results ofnorthern-blot analysis, it can be assumed that in vivo, “SGRF” is mainlyexpressed in activated T cells.

[0038] Furthermore, the culture supernatant of “SGRF” transfected CHOcells showed an activity, which supported the proliferation of bonemarrow cells (FIG. 12), in the presence of the kit ligand.

[0039] The characteristics of “SGRF” such as those above, suggest thatit is a kind of a typical interleukin. “SGRF”, as are most cytokinesisolated so far, is thought to be involved in the lymphoid andhematopoietic systems. Therefore, it can be applied as a therapeutic orpreventive drug in diseases of the lymphoid and hematopoietic systems,and also in diseases associated with defects in cell proliferation.

[0040] The protein of the present invention can be prepared by methodscommonly known to one skilled in the art, as a recombinant protein madeusing genetic engineering techniques, and also as a natural protein. Forexample, a recombinant protein can be prepared by, inserting DNAencoding the protein of the present invention (for example, DNAcomprising the nucleotide sequence in SEQ ID NO:1) into a suitableexpression vector, introducing this into a host cell, and purifying theprotein from the resulting transformant or the culture supernatant. Thenatural protein can be prepared by immobilizing( in a column, antibodiestaken from immunizing a small animal with the recombinant protein, andperforming affinity chromatography for extracts of tissues or cells (forexample, testis, lymph nodes, thymus, etc.) expressing the protein ofthe present invention.

[0041] Also, this invention features a protein, which is functionallyequivalent to the “SGRF” protein (SEQ ID NO:1). The method of insertinga mutation into the amino acids of a protein is a well-known method forisolating such proteins. In other words, for a person skilled in theart, the preparation of a protein functionally equivalent to the “SGRF”protein, is something which can be generally done using various methodssuch as the PCR-mediated, site-specific-mutation-induction system(GIBCO-BRL, Gaithersburg, Md.), oligonucleotide-mediated,site-specific-mutation-induction method (Kramer, W. and Fritz, H J(1987) Methods in Enzymol., 154:350-367) suitably replacing amino acidsin the “SGRF” protein shown in SEQ ID NO:1, which do not influence thefunction. Mutations of amino acids can occur spontaneously as well.Therefore, the protein of the invention includes those proteins that arefunctionally equivalent to the “SGRF” protein, having an amino acidsequence in which one or more amino acids in the amino acid sequence ofthe “SGRF” protein (SEQ ID NO:1) have been replaced, deleted, added,and/or inserted. The term “functionally equivalent” as used herein,refers to a protein having a cytokine activity equivalent to that of the“SGRF” protein. The cytokine activity of the “SGRF” protein includes,for example, a proliferation-supporting activity (Example 11) towardscells which are Lin negative, Sca-1 positive and c-kit positive.

[0042] The number of amino acids that are mutated is not particularlyrestricted, as long as a cytokine activity equivalent to that of the“SGRF” protein is maintained. Normally, it is within 50 amino acids,preferably within 30 amino acids, more preferably within 10 amino acidsand even more preferably within 5 amino acids. The site of mutation maybe any site, as long as a cytokine activity equivalent to that of the“SGRF” protein is maintained.

[0043] A “conservative amino acid substitution” is one in which an aminoacid residue is replaced with another residue having a chemicallysimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

[0044] In the present invention, the protein having numerous depletionsin the amino acid sequence of the “SGRF” protein (SEQ ID NO:1) includesa partial peptide. The partial peptide includes, for example, a proteinof which the signal peptide has been excluded from the “SGRF” protein ofSEQ ID NO:1.

[0045] Also, a fusion protein can be given as a protein comprising theamino acid sequence of the “SGRF” protein and several amino acids addedthereto. Fusion proteins are, for example, fusions of the abovedescribed proteins and other peptides or proteins, and are included inthe present invention. Fusion proteins can be made by techniquescommonly known to a person skilled in the art, such as linking the DNAencoding the protein of the invention and with DNA encoding otherpeptides or proteins, so as the frames match, inserting this into anexpression vector and expressing it in a host. There is no restrictionas to the peptides or proteins fused to the protein of the presentinvention.

[0046] Commonly known peptides, for example, FLAG (Hopp, T. P. et al.,Biotechnology (1988) 6:1204-1210), 6×His constituting six His(histidine) residues, 10×His, Influenza agglutinin (HA), human c-mycfragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag,SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein Cfragment can be used as peptides that are fused to the protein of thepresent invention. Examples of proteins that are fused to protein of theinvention are, GST (glutathione-S-transferase), HA (Influenzaagglutinin), Immunoglobulin constant region, β-galactosidase and MBP(maltose-binding protein). Fusion proteins can be prepared by fusingcommercially available DNA encoding these peptides or proteins with theDNA encoding the protein of the present invention and expressing thefused DNA prepared.

[0047] The hybridization technique (Sambrook, J. et al., MolecularCloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is wellknown to one skilled in the art as an alternative method for isolating aprotein functionally equivalent to the “SGRF” protein (SEQ ID NO:1). Inother words, for a person skilled in the art, it is a general procedureto prepare a protein functionally equivalent to the “SGRF” protein, byisolating DNA having a high homology with the whole or part of the DNAencoding the “SGRF” protein used as a base for the preparation of theprotein. Therefore, the protein of the present invention also includesproteins, which are functionally equivalent to the “SGRF” protein andare encoded by DNA hybridizing with DNA encoding the “SGRF” protein. Theterm “functionally equivalent” as used herein, means as mentioned above,proteins that show a cytokine activity equivalent to that of the “SGRF”protein. Apart from humans, for example, mice, rats, cows, monkeys andpigs can be used as animals from which functionally equivalent proteinscan be isolated. One skilled in the art can suitably select thestringency of hybridization for isolating DNA encoding a functionallyequivalent protein, but normally, it is equilibrium hybridization atabout 42° C., 2×SSC, 0.1% SDS (low stringency); about 50° C., 2×SSC,0.1% SDS (medium stringency); or about 65° C., 2×SSC, 0.1/% SDS (highstringency). If washings are required to reach equilibrium, then thewashings are performed using the same buffer as the originalhybridization solution, a listed above. In general, the higher thetemperature, the higher is the homology of the DNA obtainable. “Highhomology” refers to, in comparison with the amino acid sequences of the“SGRF” protein, normally a homology of 40% or higher, preferably 60% orhigher, more preferably 80% or higher, even more preferably 95% orhigher. The homology of a protein can be determined by following thealgorithm in Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA(1983) 80:726-730.

[0048] The “percent identity” of two amino acid sequences or of twonucleic acids is determined using the algorithm of Karlin and Altschul(Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlinand Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches are performed with the XBLASTprogram, score=50, wordlength=3. Where gaps exist between two sequences,Gapped BLAST is utilized as described in Altschul et al. (Nucleic AcidsRes. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

[0049] This invention also relates to a DNA encoding the above protein.There is no particular restriction as to the DNA of the presentinvention as long as it encodes the protein of the present invention andincludes cDNA, genomic DNA and chemically synthesized DNA. cDNA can beprepared by, making a primer using the nucleotide sequence of the “SGRF”cDNA, disclosed in SEQ ID NO:2, and performing RT-PCR using the mRNAprepared from cells expressing the “SGRF” protein as the template. Inthe case of genomic DNA, preparation can be done by the plaquehybridization method using a genomic DNA inserted λ phage library andthe cDNA probe obtained. The nucleotide sequence of the DNA acquired canbe decided by ordinary methods in the art using the commerciallyavailable “dye terminator sequencing kit” (Applied Biosystems). The DNAof the present invention, as stated later, can be utilized for theproduction of a recombinant protein and gene therapy.

[0050] An “isolated nucleic acid” is a nucleic acid the structure ofwhich is not identical to that of any naturally occurring nucleic acidor to that of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample, (a) a DNA which has the sequence of part of a naturallyOccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in mixtures of different (i) DNA molecules, (ii)transfected cells, or (iii) cell clones: e.g., as these occur in a DNAlibrary such as a cDNA or genomic DNA library.

[0051] The term “substantially pure” as used herein in reference to agiven polypeptide means that the polypeptide is substantially free fromother biological macromolecules. The substantially pure polypeptide isat least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight.Purity can be measured by any appropriate standard method, for example,by column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

[0052] The present invention also features a vector into which the DNAof the present invention has been inserted. There is no restriction asto the vector to which DNA is inserted, and various vectors such asthose for expressing the protein of the present invention in vivo andthose for preparing the recombinant protein can be used according to theobjective. To express the protein of the present invention in vivo(especially for gene therapy), various viral vectors and non-viralvectors can be used. Examples of viral vectors are, adenovirus vectors(pAdexLcw and such) and retrovirus vectors (pZlPneo and such).Expression vectors are especially useful when using vectors for theobjective of producing the protein of the invention. For example, whenusing Escherichia coli the “pQE vector” (Qiagen, Hilden, Germany), whenusing yeast “SP-Q01” (Stratagene, La Jolla, Calif.), when using insectcells “Bac-to-Bac baculovirus expression system” (GIBCO-BRL,Gaithersburg, Md.) are highly appropriate, but there is no restriction.Also, when using mammalian cells such as CHO cells, COS cells, NIH3T3cells, for example, the “LacSwitch II expression system (Stratagene, LaJolla, Calif.) is highly suitable, but there is no restriction.Insertion of the DNA of the present invention into a vector can be doneusing ordinary methods in the art.

[0053] The present invention also refers to a transformant, carrying, inan expressible manner, the DNA of the present invention. Thetransformant of the present invention includes, those carrying theabove-mentioned vector into which DNA of the present invention has beeninserted, and those host genomes into which the DNA of the presentinvention has been integrated. As long as the DNA of the presentinvention is maintained in an expressible manner, no distinction is madeas to the form of existence of the transformants. There is no particularrestriction as to the cells into which the vector is inserted. Whenusing the cells to express the protein of the present invention for thepurpose of gene therapy by the ex vivo method, various cells (forexample, various cells of the immune system) can be used as target cellsaccording to the type of disease. Also, when the purpose is to producethe protein of the present invention, for example, E. coli, yeast,animal cells and insect cells can be used in combination with thevectors that are utilized. Introduction of a vector into a cell can bedone using commonly known methods such as electroporation and calciumphosphate method.

[0054] The separation and purification of the recombinant protein fromthe transformant made to produce the protein can be done using ordinarymethods. The recombinant protein can be purified and prepared by, forexample, ion exchange chromatography, reverse phase chromatography, gelfiltration, or affinity chromatography where an antibody against theprotein of the present invention has been immobilized in the column, orby combining two or more of these columns. Also when expressing theprotein of the present invention inside host cells (for example, animalcells and E. coli) as a fusion protein with glutathione-S-transferaseprotein or as a recombinant protein supplemented with multiplehistidines, the expressed recombinant protein can be purified using aglutathione column or nickel column. After purifying the fusion protein,it is also possible to exclude regions other than the objective proteinby cutting with thrombin or factor-Xa as required.

[0055] The present invention also features an antibody binding to theprotein of the invention. There is no particular restriction as to theform of the antibody of the present invention and include, apart frompolyclonal antibodies, monoclonal antibodies as well. The antiserumobtained by immunizing animals such as rabbits with the protein of thepresent invention, polyclonal and monoclonal antibodies of all classes,humanized antibodies made by genetic engineering, human antibodies, arealso included., The antibodies of the present invention can be preparedby the following methods. Polyclonal antibodies can be made by,obtaining the serum of small animals such as rabbits immunized with theprotein of the present invention, attaining a fraction recognizing onlythe protein of the present invention by an affinity column coupled withthe protein of the present invention, and purifying immunoglobulin G orM from this fraction by a protein G or protein A column. Monoclonalantibodies can be made by immunizing small animals such as mice with theprotein of the present invention, excising the spleen from the animal,homogenizing the organ into cells, fusing the cells with mouse bonemarrow cells using a reagent such as polyethylene glycol, selectingclones that produce antibodies against the protein of the invention fromthe fused cells (hybridomas), transplanting the obtained hybridomas intothe abdominal cavity of a mouse, and extracting ascites. The obtainedmonoclonal antibodies can be purified by, for example, ammonium sulfateprecipitation, protein A or protein G column, DEAE ion exchangechromatography, or an affinity column to which the protein of thepresent invention is coupled. The antibody of the invention can be usedfor purifying and detecting the protein of the invention. It can also beused as a pharmaceutical drug to control the function of the presentprotein. When using the antibody as a drug for humans, in the point ofimmunogenicity, the human antibodies or the humanized antibodies areeffective. The human antibodies or humanized antibodies can be preparedby methods commonly known to one skilled in the art. For example, humanantibodies can be prepared by, immunizing a mouse whose immune systemhas been changed to that of humans, using the protein of the invention.Also, humanized antibodies can be prepared by, for example, cloning theantibody gene from monoclonal antibody producing cells and using the CDRgraft method which transplants the antigen-recognition site of the geneinto an already known human antibody.

[0056] The present invention also relates to a method for screening achemical compound that binds to the protein of the invention. Thescreening method of the invention includes the steps of, (a) exposing atest sample to the protein of the invention, (b) detecting the bindingactivity between the test sample and the protein of the invention, and(c) selecting a compound having an activity to bind to the protein ofthe invention. Any test sample call be used without particularrestrictions. Examples are, synthetic low molecular weight compoundlibraries, purified proteins, expression products of gene libraries,synthetic peptide libraries, cell extracts, and culture supernatants.Selection of a compound that has an activity to bind to the protein ofthe invention can be done using methods commonly known to one skilled inthe art.

[0057] The screening of a protein which binds to the protein of theinvention can be done by, for example, creating a cDNA library fromtissues or cells (for example, testis, lymph nodes and thymus) predictedto express a protein binding to the protein of the invention using aphage vector (λgtl1 and Zap11), expressing this cDNA library onLB-agarose, fixing the expressed proteins on the filter, biotin-labelingthe protein or the invention or purifying it as a fusion protein withGST protein, reacting this with the above-described filter, anddetecting plaques expressing the binding proteins using streptavidin oranti-GST antibody (West Western Blotting method) (Skolnik E Y, MargolisB, Mohammadi M, Lowenstein E, Fischer R, Drepps A, Ullrich A, andSchlessinger J (1991) Cloning of Pl3 kinase-associated p85 utilizing anovel method for expression/cloning of target proteins for receptortyrosine kinases. Cell 65:83-90).

[0058] The screening of a protein binding to the protein of theinvention or the gene of the protein, can also be done by following “thetwo hybrid system” (“MATCHMAKER Two-hybrid System”, “MammalianMATCHMAKER Two-hybrid Assay Kit”, “MATCHMAKER One-Hybrid System”(Clontech), “HybriZAP Two-Hybrid Vector System” (Stratagene), Reference,“Dalton, S. and Treisman, R. (1992) Characterization of SAP-l, a proteinrecruited by serum response factor to the c-fos serum response element.Cell 68:597-612”). Namely, the protein of the invention is fused to theSRF binding region or GAL4 binding region and expressed in yeast cells.A cDNA library, is prepared from cells predicted to express a proteinbinding to the protein of the invention so as to express the ligandfused to the VP16 or GaL4 transcriptional activation region. The cDNAlibrary is then introduced into the above yeast cells and the cDNAderived from the library is isolated from the positive clones detected(when a protein binding to the protein of the invention is expressed inyeast cells, the binding of the two activates a reporter gene makingpositive clones detectable). The isolated cDNA is expressed byintroducing it into E. coli to obtain a protein encoded by the cDNA.Furthermore, a protein binding to the protein of the invention can bescreened by, applying the culture supernatants or cell extracts of cellspredicted to express a protein binding to the protein of the inventiononto an affinity column in which the protein of the invention isimmobilized and purifying the protein that binds specifically to thecolumn.

[0059] Also, the method of screening molecules which bind when theimmobilized protein of the invention is exposed to synthetic chemicalcompounds, or natural substance banks, or a random phase peptide displaylibrary, or the method of screening using high-thoughput based oncombinatorial chemistry techniques (Wrighton N C; Farrel F X; Chang R;Kashyap A K; Barbone F P; Mulcahy L S; Johnson D L; Barret R W; JolliffeL K; Dower W J. Small peptides as potent mimetics of the protein hormoneerythropoietin, Science (UNITED STATES) Jul. 26 1996, 273 p458-68,Verdine G L., The combinatorial chemistry of nature. Nature (ENGLAND)Nov. 7 1996, 384 p11-13, Hogan J C Jr., Directed combinatorial chemistryNature (ENGLAND) Nov. 7 1996, 384 p17-9) to isolate low molecular weightcompounds, proteins (or the genes) and peptides are methods well knownto one skilled in the art.

[0060] The present invention also relates to a method for screening acompound able to promote or inhibit the activity of the protein of theinvention. It was found that the protein of the invention has aproliferation-supporting activity for bone marrow cells in the presenceof the kit ligand. Therefore, using this activity as an indicator,screening of a compound able to promote or inhibit activity of theprotein of the invention can be performed. Namely, this screening can bedone using the method comprising the steps of: (a) exposing the proteinof the invention and the kit ligand to mammalian bone marrow cells underthe presence of a test compound; (b) detecting the proliferation of thebone marrow cells; and (c) selecting a compound which promotes orinhibits the proliferation of bone marrow cells when compared with theassay in the absence of a test sample (control).

[0061] There are no particular restrictions as to the test sample used.Examples are, libraries of synthetic low molecular compounds, purifiedproteins, expression products of gene libraries, synthetic peptidelibraries, cell extracts and culture supernatants. The compound isolatedby the above-described screening of a protein binding to the protein ofthe invention may also be used as a test compound.

[0062] The protein of the present invention and the kit ligand may berecombinant or natural proteins. Also, as long as the activity ismaintained, may be a partial peptide. The kit ligand may also be acommercially available one.

[0063] Lin negative, Sca-1 positive and c-kit positive bone marrow cellsare preferred for the screening. Bone marrow cells that are additionallyCD34 positive are preferred more.

[0064] The culture conditions and detection of proliferation of bonemarrow cells can be done, for example, in conformance with Example 11.

[0065] As a result of the detection, compared with the proliferation ofbone marrow cells ill the absence of the test compound (control), if theproliferation of bone marrow cells is suppressed with the addition of atest compound, then the test compound is judged to be a compound (orincludes the compound), which inhibits the activity of the protein ofthe invention. On the other hand, if the proliferation is promoted bythe addition of the test compound (or includes the compound), it isjudged to be a compound that promotes the activity of the protein of theinvention.

[0066] The protein of the present invention can be used as a reagent inresearch to control the proliferation of cells of the lymphoid andhematopoietic systems. The compound isolated by the above-mentionedscreening, can be used as an inhibitor or promoter of the protein in theinvention. Moreover, the protein of the invention or these compounds canalso be utilized as drugs for the prevention and therapy of diseasesassociated with defects in cell proliferation and lymphoid andhematopoietic systems.

[0067] When using the protein of the invention or a compound thatcontrols the activity of the protein as drugs, they can be formulatedinto a dosage formulated using commonly known pharmaceutical preparationmethods. For example, according to the need, the drugs can be takenorally (as sugar-coated tablets, capsules, elixirs and microcapsules) ornon-orally (such as, percutaneously, intranasally, bronchially,intramuscularly and intravenously) in the form of injections of sterilesolutions or suspensions with water or any other pharmaceuticallyacceptable liquid. For example, the protein of the invention orcompounds controlling the activity of the protein can be mixed withphysiologically acceptable carriers, flavoring agents, excipients,vehicles, preservatives, stabilizers and binders, in a unit dose formrequired for generally accepted drug implementation. The amount ofactive ingredients in these preparations makes a suitable dosage withinthe indicated range acquirable.

[0068] Examples for additives which can be mixed to tablets and capsulesare, hinders such as gelatin, corn starch, tragacanth gum and arabicgum; excipients such as crystalline cellulose; swelling agents such ascornstarch, gelatin and alginic acid; lubricators such as magnesiumstearate; sweeteners such as sucrose, lactose or saccharin; flavoringagents such as peppermint, Gaultheria adeuothrix oil and cherry. Whenthe unit dosage form is a capsule, a liquid carrier, such as oil, canalso be included in the above ingredients. Sterile composites forinjections can be formulated following normal drug implementations usingvehicles such as distilled water used for injections.

[0069] Physiological saline, glucose, and other isotonic liquidsincluding adjuvants, such as D-sorbitol, D-mannose, D-mannitol, andsodium chloride, can be used as aqueous Solutions for injections. Thesecan be used in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, non-ionic surfactants, such as Polysorbate 80 (TM) andHCO-50.

[0070] Sesame oil or soy-bean oil can be used as a oleaginous liquid andmay be used in conjunction with benzyl benzoate or benzyl alcohol as asolubilizer; may be formulated with a buffer such as phosphate andsodium acetate; a pain-killer such as procaine hydrochloride; astabilizer such as benzyl alcohol, phenol, and an anti-oxidant. Theprepared injection is filled into a suitable ampule.

[0071] One skilled in the art can suitably select the dosage and methodof administration according to the body-weight, age, and symptoms of apatient.

[0072] For example, although there are some differences according to thepatient, target organ, symptoms and method of administration, the doseis about 1 μg to about 100 mg per day for a normal adult (weight 60 kg)when the protein is given as an injection.

[0073] For a compound controlling the activity of the protein of theinvention, although it can vary according to the symptoms, the dosage isabout 0.1 to about 100 mg per day, preferably about 0.1 to about 50 mgper day and more preferably about 0.1 to about 20 mg per day, whenadministered orally.

[0074] When administering non-orally in the form of an injection to anormal adult (weight 60 kg), although there are some differencesaccording to the patient, target organ, symptoms and method ofadministration, it is convenient to intravenously inject a dose of about0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg perday and more preferably about 0.1 to about 10 mg per day. Also, in thecase of other animals too, it is possible to administer an amountconverted to 60 kg of body-weight.

[0075] This invention also features a DNA containing at least 15nucleotides, which can specifically hybridize with DNA encoding the“SGRF” protein. The term “specifically hybridize” as used herein,indicates that cross-hybridization does not occur significantly with DNAencoding other proteins, in the above-mentioned hybridizing conditions,preferably under stringent hybridizing conditions.

[0076] Such DNA can be utilized to detect DNA encoding the “SGRF”protein, as an isolation probe, and also as a primer for amplification.Specifically, the primers in SEQ ID NOs:3 to 20 can be given asexamples. Such DNA can also be used as an oligo nucleotide or aribozyme.

[0077] An antisense oligonucleotide is preferably an antisenseoligonucleotide against at least 15 continuous nucleotides in thenucleotide sequence of SEQ ID NO:2. The above-mentioned antisenseoligonucleotide, which contains an initiation codon in theabove-mentioned at least 15 continuous nucleotides, is even morepreferred.

[0078] Derivatives or modified products of antisense oligonucleotidescan be used as antisense oligonucleotides. Examples are, lower classalkyl phosphate modifications such as methyl-phosphonate-type orethyl-phosphonate-type and phosphothioate or phosphoramidate.

[0079] The term “antisense oligonucleotides” as used herein means, notonly those in which the nucleotides corresponding to those constitutinga specified region of a DNA or nRNA are complementary, but also thosehaving a mismatch of one or more nucleotides, as long as DNA or mRNA andan oligonucleotide can specifically hybridize with the nucleotidesequence of SEQ ID NO:2.

[0080] Such DNAs are indicated as those having, in the “at least 15continuous nucleotide sequence region”, a homology of at least 70% orhigher, preferably at 80% or higher, more preferably 90% or higher, evenmore preferably 95% or higher to the nucleotide sequence of SEQ ID NO:2.The algorithm stated herein can be used to determine homology.

[0081] The antisense oligonucleotide derivative of the presentinvention, acts upon cells producing the protein of the invention bybinding to the DNA or mRNA encoding the protein and inhibits itstranscription or translation, promotes the degradation of the mRNA,inhibiting the expression of the protein of the invention resulting inthe inhibition of the protein's function.

[0082] The antisense oligonucleotide derivative of the present inventioncan be made into an external preparation such as a liniment and apoultice by mixing with a suitable base material, which is inactiveagainst the derivatives.

[0083] Also, as needed, the derivatives can be formulated into tablets,powders, granules, capsules, liposome capsules, injections, solutions,nose-drops and freeze-drying agents by adding excipients, isotonicagents, dissolving auxiliaries, stabilizers, preservatives andpain-killers. These can be prepared using usual methods.

[0084] The antisense oligonucleotide derivative is given to the patientby, directly applying onto the ailing site or by injecting into a bloodvessel so that it will reach the site of ailment. An antisense-mountingmedium can also be used to increase durability andmembrane-permeability. Examples are, liposome, poly-L lysine, lipid,cholesterol, lipofectin or derivatives of these.

[0085] The dosage of the antisense oligonucleotide derivative of thepresent invention can be adjusted suitably according to the patient'scondition and used in desired amounts. For example, a dose range of 0.1to 100 mg/kg, preferably 0.1 to 50 mg/kg can be administered.

BRIEF DESCRIPTION OF THE DRAWINGS

[0086]FIG. 1 shows the “SGRF” cDNA nucleotide sequence together with thededuced amino acid sequence. The mRNA destabilizing sequence isunderlined.

[0087]FIG. 2 shows the alignment of the consensus sequence of “SGRF” andproteins belonging to the IL-6/G-CSF/MGF family. The consensus sequenceof this family is thought to be C-x(9)-C-x(6)-G-1-x(2)-[FY]- x(3)-L, iswell preserved in “SGRF” as well, excluding the point that the number ofthe first x is 11 instead of 9. In the figure, Sooty Mangabey is ananimal of the Cercocebus species and Rhesus Macaque is of the RhesusMonkey species.

[0088]FIG. 3 shows the hydrophobicity of “SGRF”.

[0089]FIG. 4 shows the electrophoretic pattern of the “SGRF” expressionin normal human tissue as detected by Northern blot analysis. Markersare, from the left, 9.5 kb, 7.5 kb, 4.4 kb, 2.4 kb, and 1.35 kb.

[0090]FIG. 5 shows the electrophoretic pattern of the “SGRF” expressionin human fetal tissue and tumor cells as detected by Northern blotanalysis. Markers are, from the left, 9.5 kb, 7.5 kb, 4.4 kb, 2.4 kb,and 1.35 kb.

[0091]FIG. 6 shows the amino acid sequence of a protein presumed to bethe genomic DNA nucleotide sequence of SGRF. Introns are shown in simpleletters, exons in capitals. Sequences expected to be those of the TATABox and poly A addition signal are underlined.

[0092]FIG. 7 shows the result of PCR analysis using NIGMS human/rodentsomatic cell hybrid mapping panel #2. The numbers show the humanchromosomes contained in the hybridoma DNA.

shows human (female) genomic DNA, M shows mouse genomic DNA. 100 bpladder has been used as a marker. The band seen around 200 bp is thoughtto be a non-specific background derived from mouse chromosomes.

[0093]FIG. 8 shows the pCHO-SGRFg map.

[0094]FIG. 9 shows the pCHO-SGRF map.

[0095]FIG. 10 shows the pLG-SGRF map.

[0096]FIG. 11 shows the pLG-SGRFg map.

[0097]FIG. 12 shows the effect of the SGRF-gene-inserted, CHO cellculture supernatant on the proliferation of bone marrow cells.

DETAILED DESCRIPTION OF THE INVENTION

[0098] The present invention will be explained in detail below withreference to examples, but it is not limited thereto.

EXAMPLE 1

[0099] Isolation of the “SGRF” Gene

[0100] When the GenBank EST database was searched by means of TBLASTusing the human G-CSF protein sequence, the EST of Reg. No: AA418955showed a weak homology to G-CSF. Based on this sequence, when an ESTsequence considered to be reading the same gene was searched, four otherregistered ESTs (AA418747, U38443, AA729815, AA418955) were found. Thesesequences were aligned using DNASIS, the consensus sequence wasextracted, and the following primers were designed:“ILX-1”(GAGAAGAGGGAGATGAAGAGACTAC;/SEQ.ID.NO:3)“ILX-2”(CTGAGTCCTTGGGGGTCACAGCCAT;/SEQ.ID.NO:4)“ILX-3”(GTGGGACCTGCATATGTTGAAAATT;/SEQ.ID.NO:5“ILX-4”(CCCCAAATTTCCCTTCCCATCTAATA;/SEQ.ID.NO:6)“ILX-5”(CCCTACTGGGCCTCAGCCAACTCCT;/SEQ.ID.NO:7) and“ILX-6”(GGAGCAGAGAAGGCTCCCCTGTGAA./SEQ.ID.NO:8

[0101] Using the human fetal spleen library (Marathon-Ready cDNA;Clontech), sequential PCR was performed in combinations of primersstated below, divided into 3 fragments, and amplified separately(5′side, central area, and 3′side). The primers used for the 5′sideamplification were, “AP1” (Clontech) and “ILX-6” in the primary PCR,“AP2” (Clontech) and “ILX-2” in the nested PCR. As to the central area,“ILX-1” and “ILX-4” were used for the primary and nested PCRs. For the3′ side, “AP1” (Clontech) and “ILX-5” were used in the primary PCR,“AP2” (Clontech) and “ILX-3” in the nested PCR. Amplifications by boththe primary and nested PCRs were done under conditions in which thoserecommended by the Manufacturer were partially changed (touchdown PCR: 1min at 96° C., following 30 sec at 96° C., 5 cycles of “4 min at 72°C.”, following 30 sec at 96° C., 5 cycles of “4 min at 70° C.”,following 20 sec at 96° C. and 26 cycles of “4 mil at 68° C.”. However,TaKaRa Ex Taq (Takara Shuzo) and attached buffers were used instead ofAdvantage Klentaq Polyerase Mix). The obtained DNA were thenelectrophoresed on agarose gel, the corresponding bands were cut-off,after purifying by QIAEX II Gel Extraction Kit (QIAGEN), were clonedinto the plasmid pT7Blue (R) T-vector (Novagen). The obtained plasmidswere named pT7Blue-ILX1-4 (the vector which cloned the central area),pT7Blue-ILX5′ (the vector which cloned the 5′end area), andpT7Blue-ILX3′ (the vector which cloned the 3′end area), respectively.Each nucleotide sequence cloned was determined using ABI PRISM DyeTerminator Cycle Sequencing Ready Reaction Kit with Amplitaq DNAPolymerase FS and 377 A DNA Sequencer (Perkin-elmer).

[0102] As a result, the full length of the isolated cDNA was 1 Kb, andencoded a protein comprising 189 amino acids (FIG. 1). This protein, notonly had a consensus sequence typical of IL-6/G-CSF/MGF family (FIG. 2),but also a hydrophobic region considered to be a signal peptide in the Nterminal (FIG. 3). Also, the binding site of N-type sugar-chain was notseen. In the 3′ non-coding region, four mRNA destabilizing sequencescalled ARE (AT Rich element), often seen in cytokine mRNAs (FIG. 1),were detected, having characteristics of a typical cytokine. Based onthe structural homology, this molecule was named “SGRF”(Interleukin-Six, G-CSF Related Factor).

EXAMPLE 2

[0103] Northern Blot Analysis of “SGRF” Expression

[0104] A 500 bp fragment obtained by treating pT7Blue-ILX1-4 with BamH1was used as the probe of “SGRF”. The above probe was “□-32P”dCTP labeledby random priming method using Ready-to Go DNA labeling beads(Pharmacia), and hybridization was done according to methods recommendedby the Manufacturer within the ExpressHyb Hybridization Solution(Clontech) against the Multiple Tissue Northern Blot (Human, Human III,Human IV, Human Fetal II, Human Cancer Cell Line (Clontech) Filters. Asa result, in normal tissues, “SGRF” was mainly expressed in the testisand lymph nodes, and an mRNA of approximately I kb was detected. Anextremely low expression was Found in the thymus. However, since a longautoradiography (1 week) was required For the detection of these bands,the expression level in these are considered to be low. “SGRF” mRNA wasnot in a detectable level in other tissues.

[0105] When cancer cell lines were analyzed, very strong levels ofexpression were detected in two cell lines; K(562 (Chronic MyelogenousLeukemia) and SW480 (Colorectal Adenocarcinoma) (FIG. 5). In the othercell lines, “SGRF” mRNA was not in a detectable level.

EXAMPLE 3

[0106] Construction of the “SGRF” Expression Vector

[0107] Two primers, “ILXATG” TTGAATTCCACCATGCTGGGGAGCAGAGCTGT/SEQ IDNO:14), “ILXTAA” (AAAGATCTTAGGGACTCAGGGTTGCTGC/SEQ ID NO:15), wereconstructed, a gene consisting all the coding regions reconstituted bypT7Blue-1 LX 1-4 and pT7Blue-ILX5′, and introduced into an animal cellexpression vector. Namely, the band amplified using pT7Blue-ILX5′as thetemplate and “ILX-2” and “ILXATG” as primers, and the band amplifiedusing pT7Blue-ILX1-4 as the template and “ILX-1” and “ILXTAA” asprimers, were mixed in equal amounts, and re-amplification was doneusing this as the new template with the primers “ILXATG” and “ILXTAA”.The resulting band was treated with restriction enzymes EcoRI and BamHI,and cloned into the EcoRT, Bgl II site of all animal cell expressionplasmid pCOS1 to create pCOS-SGRF. TaKaRa ExTaq (Takara Shuzo) was usedfor DNA amplification for 20 cycles of 30 sec at 96° C., following 40sec at 60° C., and following I min 20 sec at 72° C.

EXAMPLE 4

[0108] The Polyclonal Antibody Corresponding to “SGRF”

[0109] Two kinds of partial peptides of “SGRF” (GGSSPAWTQCQQLSQ/24-38position of the amino acid sequence of SEQ ID NO:1, GDGCDPQGLRDNSQF,74-88 position of the amino acid sequence of SEQ ID NO:1) werechemically synthesized. Two rabbits were immunized with these,respectively, to obtain polyclonal antibodies (Sawady). The respectiveantibodies were affinity-purified using respective peptide columns. Thefollowing analyses were done using one of the antibodies against thepeptide of SGRF (24-38 position of the amino acid sequence of SEQ IDNO:1).

[0110] Alkyl phosphatase-binding, mouse-anti-rabbit IgG antibody andalkyl phosphatase substrate were used for detection.

EXAMPLE 5

[0111] Genomic DNA of SGRF

[0112] The following sequences were prepared and used for the analysisof the promoter regions-5′non translating region, translating region, 3′non-translating region from the genomic DNA library. (SEQ ID NO:3) ILX-15′-GAGAAGAGGGAGATGAAGAAGACTAC-3′ (SEQ ID NO:4) ILX-25′-CTGAGTCCTTGGGGGTCACAGCCAT-3′ (SEQ ID NO:5) ILX-35′-GTGGGACCTGCATATGTTGAAAATT-3′ (SEQ ID NO:6) ILX-45′-CCCCAAATTTCCCTTCCCATCTAATA-3′ (SEQ ID NO:7) ILX-55′-CCCTACTGGGCCTCAGCCAACTCCT-3′ (SEQ ID NO:8) ILX-65′-GGAGCAGAGAAGGCTCCCCTGTGAA-3′ (SEQ ID NO:9) ILX-75′-GGGCAGAGATTCCAGGAGGACTGGT-3′ (SEQ ID NO:10) ILX-85′-CCAGTCCTGGTGGAATCTCTGCCCA-3′ (SEQ ID NO:11) ILX-95′-GAAGCTCTGCACACTGGCCTGGAGT-3′ (SEQ ID NO:12) ILX-105′-CACTCCAGGCCAGTGTGTGCAGAGCTT-3′ (SEQ ID NO:13) ILX-115′-CTGAAGGGCTATGGTGGAGAA-3′ (SEQ ID NO:14) ILX-ATG5′-TTGAATTCCACCATGCTGGGGAGCAGAGCTGT-3′ (SEQ ID NO:15) ILX-TAA5′-AAAGATCTTAGGGACTCAGGGTTGCTGC-3′ (SEQ ID NO:16) ILX-TAAECO5′-AAGAATTGTAGGGACTCAGGGTTGCTGC-3′ (SEQ ID NO:17)SGRFg5′5′-GGTTTAAATATTTGTTCTCCCTTACCCC-3′ (SEQ ID NO:18) SGRFg375′-TTCAGCTGCTTGGGAGGCTGAGGCAGG-3′ (SEQ ID NO:19) SGRFg5′-25′-AGGAATTCCACCAGGACTGGTGCAAGGCGCA-3′ (SEQ ID NO:20) SGRFg3′-25′GTCTCGAGAAAATATCATTCTCCACCATCGCCCT-3′

[0113] Genomic DNA was amplified by PCR using, for the translationregion, the above mentioned ILX-ATG primer and ILX-TAA primer and humangenomic DNA (Clontech) as the template, resulting in a band amplified toapproximately 1.5 kb. After treating this fragment with restrictionenzymes EcoRI and BglII, cloning was done by inserting into theEcoRI-BamHI site of the CHO expression plasmid pCHO1. The nucleotidesequence or the vector obtained (pCHO-SGRFg) (FIG. 8) was analyzed usingthe primers described above. As a result, it was revealed to be the SGRFgene including 3 introns.

[0114] The amplifications of the 5′ non-translating region containingthe promoter, and the 3′ non-translating region, were done using GenomeWalker Kit as the template (Clontech), the attached AP1 and AP2 primersand the above-mentioned synthetic primers according to methodsrecommended by the Manufacturer.

[0115] First, for the 5′ non-translating region, the 1^(st) PCR was doneusing Dra1 library as the template with AP1 and ILX-10 primers. Then,the 2^(nd) PCR was done with the AP2 and ILX-8 primers to obtain a bandof approximately 400 bp.

[0116] For the 3′ non-translating region, the 1^(st) PCR was done usingPvuII library as the template with AP1 and ILX-5 primers, the 2^(nd) PCRwith the AP2 and ILX-3 primers to obtain a band of app 800 bp.

[0117] The bands obtained were cut off from the agarose gel, and afterpurification , sequencing was done using 377 A DNA Sequencer(Perkin-Elmer). The genomic DNA sequence of SGRF (SEQ ID NO:21) is shownin FIG. 6 together with the deduced amino acids.

[0118] Also, NIGMS human/rodent somatic cell hybrid mapping panel #2 andGeneBridge 4 Radiation Hybrid Panel (Research Genetics) were analyzed byPCR using ILX-1 and Ilx-6 primers to examine the chromosome location. Asa result, from NIGMS human/rodent somatic cell hybrid mapping panel #2analysis, it was revealed that the SGRF gene exists on the 12^(th)chromosome (FIG. 7).

[0119] The analysis from GeneBridge 4 Radiation Hybrid Panel, revealedthat SGRF exists in 12q13 and is, Chromosome Chr12, Places 8.77 cR fromWJ-7107 (lod>3.0).

EXAMPLE 6

[0120] Establishment of a CHO Cell Line Expressing SGRF

[0121] Similarly to pCOS-SGRF described in Example 3, DNA fragment ofSGRF encoding region was prepared, was cloned to the EcoRI, BamHI siteof the animal cell expression plasmid pCHO1 to create pCHO-SGRF (FIG.9). PCHO-SGRF was then transfected into CHO cells by calcium phosphatemethod and gene-introduced cells were selected in alpha-MEM culturemedium, which does not contain nucleotides. The culture supernatant wasanalyzed by SDS-PAGE and Western blotting using rabbit-polyclonalantibody.

[0122] As a result, a band with a molecular weight of about 20,000 wasdetected only in the culture supernatant of CHO cells having thisvector.

[0123] Thereafter, the MTX concentration was increased sequentially to20 nM, 100 nM and so on, and the gene was amplified while verifying theexpression to establish a CHO cell strain, which constitutively secretesSGRF. This cell strain has been deposited in the depository institutiongiven below.

[0124] (a) Name and address of the Depository Institution

[0125] Name: National Institute of BioScience and Human-TechnologyAgency of Industrial Science and Technology

[0126] Address: 1-3,Higashi-1-chome, Tsukaba-shi, Ibaraki 305-8566,Japan.

[0127] (b) Date of deposit (date of original deposit): April 9, 1999.

[0128] (c) Accession Number: FERM BP-6699

EXAMPLE 7

[0129] Purification of SGRF

[0130] In order to review purification of SGRF, producing cells(CHO-SGRF 16-5 strain) proliferated to a confluent state, were rinsedwith PBS, the medium was changed to a serum-free culture medium ASF104(AJINOMOTO), cultured for 3 to 4 clays, and the culture supernatant wascollected after filtering.

[0131] A 30 ml column was prepared using Phenyl-Sepharose HP (AmershamPharmacia Biotech), equilibrated by 10 mM Tris pH 7.5, 100 mM NaCl, andthe culture supernatant of the above mentioned CHO-SGRF16-5 straincultured in ASF medium, was 1.5 times diluted using 10 mM Tris pH 7.5and applied onto the column. After washing well with the equilibratingbuffer, extraction was done with the same buffer containing 0.1%/Tween20. The extracted solution was applied to a DEAE-Sepharose FF columnequilibrated with 10 mM Tris pH 7.5, 100 mM NaCl, washed well with theequilibrating buffer, and extracted using 10 mM Tris pH 7.5, 300 mMNaCl, recovering most of the SGRF. The extracted sample was according tonormal methods by SDS-PAGE analysis, Western blot analysis as a crudelypurified product. As a result, a band binding to the polyclonal antibodywas detected at the site of a molecular weight of around 20,000 whichwas concentrated enough lo be detected by Silver-staining andCoomassie-staining.

[0132] This crudely purified SRF protein was blotted onto a PVDFmembrane, stained with Coomassie blue, and a band with a molecularweight of about 20,000 was cut off to determine the N-terminal aminoacid sequence using Model 492 protein sequencer (Applied Biosystems). Asa result, the sequence was found to be X-Ala-Val-Pro-Gly-Gly-Ser. Thismatched the SGRF sequence of 20th Arg from the N-terminal to the 29thSer, and the signal peptide was found to be cleaved between the 19th GLYand the 20th Arg.

[0133] From the above results, the mature protein of SGRF was calculatedto be having 170 amino acids with a presumed molecular weight of 18,676and an expected isoelectric point of 5.84.

EXAMPLE 8

[0134] SGFR Vector for the Creation of a Transgenic Mouse

[0135] SGRF cDNA was amplified from pCHO-SGRF using the primers ILX-ATGand ILX-TAAECO, cleaved with the restriction enzyme EcoRI, then insertedinto the EcoRI site of transgenic expression plasmid pLG1 to createpLG-SGRF (FIG. 10).

[0136] Also, the region containing SGRF genomic DNA was amplified fromhuman genomic DNA(clontech) using primers SGRF-5′_(—)2 and SGRF-3′_(—)2,treated with the restriction enzyme EcoRI, and then inserted into theEcoRI-XhoI site of the plasmid pLG1 to create pLG-SGRFg (FIG. 11).

EXAMPLE 9

[0137] Production of the Monoclonal Antibody

[0138] Five 8-week male Balb/c mice are immunized 2 mg/head withaluminum hydroxide gel as the adjuvant, and 20 μg/head of the abovementioned SGRF protein or the partial peptide as the antigen byinjection into the peritoneal cavity. Re-immunization is done six timesin every 2 weeks by injecting 20 μg/head of the above mentioned SGRFprotein or the partial peptide. After the 3^(rd) immunization, blood isdrawn from the eye-ground venous plexus and anti SGRF antibody titer inthe serum is examined.

[0139] Three days after the final immunization, spleen cells areprepared from mice, an(l used for cell fusion. 1×108 splenocytes fromthe immunized mice washed well with MEM (Nissui Pharmaceuticals), andmurine myeloma P3-U 1×108 are mixed and centrifuged for 5 min at 1000rpm. 2 g Polyethylene glycol-1500 (PEG-1500), and 2 ml MEM are addedwhile mixing well at 37° C. and centrifuged after I mini at 600 rpm for5 mil. Further, 5 ml HBSS solution and 5 ml 20% FBS/MEM solution areadded calmly, cells are suspended well, and centrifuged at 1000 rpmafter 1 min, and the culture supernatant is discarded. The cells arere-suspended by adding 5 ml HAT medium (10-4M hypoxanthine, 4×10-7Maminopterin, and 1.5×10-5M thymidine supplemented medium). The cellsuspension is seeded in I ml/well into a 24-well culture plate (Nunc),and cultivated for 24 hr in a CO₂ incubator at 37° C., 5% CO₂, 95% air.1 mil/well HAT medium is added and cultured further for another 24 hr.Then, 1 ml of culture supernatant is discarded, I ml HAT medium is newlyadded and cultivated further for another 12 days.

[0140] For those wells in which colonized fusion cells can be detected,I ml culture supernatant is discarded, 1 ml HAT medium(aminopterin-excluded, above-mentioned HAT medium) added and cultured at37° C. Exchange of the HAT medium is similarly done for the next twodays and after culturing for 4 days, a portion of the culturesupernatant is collected to measure the anti-SGPF antibody titer byELISA.

[0141] For wells in which the antibody titer was detected, cloning isperformed by limiting dilution twice more, and clones for which a stableantibody titer was detected, are selected as hybridomas producinganti-SGRF monoclonal antibody.

EXAMPLE 10 ELISA Method

[0142] 50 μl/well of SGRF protein solution or the partial peptidesolution is seeded into a 96-well culture plate (Immunoplate, Nunc)andis left to stand at room temperature for 2 hours to coat the antigenonto the bottom of the plate-well. Then, 200 μl/well of 10% FCS/PBS isadded and left to stand at room temperature for 30 min. Theabove-mentioned plate is washed 3 times with PBS, serially diluted testsample (mouse serum, hybridomas culture supernatant, monoclonal antibodyand such) is seeded in 50 μl/well, and left to stand at room temperaturefor 2 hours. Then, the plate is washed 3 times with PBS, and 100times-diluted peroxidase-binding goat-anti-mouse IgG antibody is seededin 50 μl/well as the secondary antigen and left to stand at roomtemperature for 2 hours. After washing with PBS, 200 μl/well ofperoxidase substrate (1% hydrogen peroxide, 0.1M acetic acicl-0.05Mphosphate buffer, 2 mM 2,2′-azino-di-3-ethyl-benzothiazine sulfate) isadded and after leaving at room temperature for 10 to 30 min, theantibody titer is calculated using calorimetry at 414 nm.

EXAMPLE 11

[0143] Bone marrow cells were prepared by extracting the thigh-bone andshank-bone of 8 to 15 week C57BL/6 male mice (CLEAR JAPAN). Aftersuspending in Nycodenz (Nycomed Pharm AS), the specific gravity wasadjusted to 1.063, stratified to NycoPrep 1.077 Animal (Nycomed PharmAS) and centrifuged for 30 min at 2300 rpm (Hitachi, 05PR22), 20° C. Theintermediate layer was collected, suspended in FACS buffer (2% fetalBovine Serum (FBS, Moregate) containing Dulbecco's phosphate buffer),was collected by centrifuging for 10 min at 1500 rpm, after which 1 μgof biotin-labeled anti-Mac-1 antibody, biotin-labeled anti-Gr-1antibody, biotin-labeled anti-TER119 antibody, biotin-labeled anti-CD3εantibody, and biotin-labeled B220 antibody (all by Pharmingen) wereadded per 1×106 cells. After leaving aside for 30 min on ice, was washedwith FACS buffer, 1 μl avidin-labeled microbeads (10 Beads Avidin,ImmunoTech) were added and left to stand for 15 min oil ice. Beads werethen excluded by a magnetic holder-, the floating cells were collectedby centrifuging for I min (Tomy MRX-150) at 5000 rpm. After discardingthe supernatant, 1 ρg per 1×106 cells of FITC-labeled anti-CD34antibody, PE-labeled anti Sca-1 antibody, APC-labeled c-kit antibody(all 3 by Pharmingen), RED613-lebeled streptavidin (LifeTech Oriental)were added and reacted for 30 min on ice. After washing with FACSbuffer, was suspended in 1 ml FACS buffer for 1×10⁶ cells, andfractionated by EPICS ELITE (Beckman Coulter). The definitions of therespective fractions are as follows:

[0144] RED613-negative PE-negative APC-positive=Lin(−) Sca-1(−) c-kit(+)fraction

[0145] RED613-negative PE-positive APC-positive=Lin(−) Sca-1(+)c-kit(+)fraction

[0146] RED613-negative PE-positive APC-positive FITC-positive=Lin(−)Sca-1(−) c-kit(+) CD34(+) fraction

[0147] RED6 13-negative PE-positive APC-positive FITC-negative=Lin(−)Sca-l(+) c-kit(+) CD34(−) fraction

[0148] The obtained cell fractions were diluted by the Iscove's modified DDulbecco's medium (10% FBS/IMDM) so that there were 10,000, 2000,and 400 cells per I ml of medium and were seeded in 50 ml into a 96-wellculture plate.

[0149] To this, 50 μl of, (1) medium only (10% FBS/IMDM), (2) medium towhich mock has been diluted to 20% (mock), (3) SGRF-expressed CHOculture supernatant (SGRF), (4) medium to which mouse kit ligand 10ng/ml has been added (KL), (5) medium to which 20% mock, 10 ng/ml mousekit ligand have been added (mock +ILL), (6) SGRF-expressed CHO culturesupernatant to which 10 ng/ml mouse kit ligand has been added (SGRF+KL),(7) medium to which 10 ng/ml mouse kit ligand, 5 ug/ml IL-1 I has beenadded (KL+IL-11, were supplemented and then cultured for 10 days at 37°C., 5% CO₂.

[0150] Also, as for (1) and (3) mentioned above, 10 ng/ml, 1 ng/ml mousekit ligand IL-11 were added respectively to prepare a similarly culturedlot as well (medium/expand, mock/expand, SGRF/expand, respectively).

[0151] After completion of culture, cell number was detected using aCell proliferation Assay Kit (Promega) as measured by Microplate ReaderModel 3550 (Bio-Rad) for the absorbance at 490 nm (FIG. 12).

[0152] As a result, although SGRF-alone showed no proliferationsupporting activity towards Lin negative, Sca-I positive and c-kitpositive cells, such a proliferation supporting activity was shown underthe presence of the mouse kit ligand. This activity was stronger in CD34positive cells. Also, if the mouse kit ligand did not exist at theinitial stages of culture, cells did not proliferate even when theligand was supplemented afterwards. From this fact it can be assumedthat SGRF does not have an activity to support stem cells.

INDUSTRIAL APPLICABILITY

[0153] The present invention provided a novel cytokine-like protein andthe DNA encoding the protein. Furthermore, a vector into which the DNAis inserted, a transformant possessing said DNA and the methods ofproduction of a recombinant protein are provided. A compound that bindsto the protein and a screening method for a compound that regulates itsactivity are also provided.

[0154] Since the protein of the invention and the gene, alike othercytokines, are believed to be associated with the activity or cellproliferation and differentiation of cells of the immune andhematopoietic systems, the use of a compound that controls said proteinis anticipated in diseases relating to the immune or hematopoieticsystems and defects in cell proliferation. A number of embodiments ofthe invention have been described. Nevertheless, it will be understoodthat various modifications may be made without departing from the spiritand scope of the invention.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 46 <210> SEQ ID NO 1<211> LENGTH: 189 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 1 Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu Leu Pro TrpThr 1 5 10 15 Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser Pro Ala TrpThr Gln 20 25 30 Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu Ala Trp SerAla His 35 40 45 Pro Leu Val Gly His Met Asp Leu Arg Glu Glu Gly Asp GluGlu Thr 50 55 60 Thr Asn Asp Val Pro His Ile Gln Cys Gly Asp Gly Cys AspPro Gln 65 70 75 80 Gly Leu Arg Asp Asn Ser Gln Phe Cys Leu Gln Arg IleHis Gln Gly 85 90 95 Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser Asp Ile PheThr Gly Glu 100 105 110 Pro Ser Leu Leu Pro Asp Ser Pro Val Gly Gln LeuHis Ala Ser Leu 115 120 125 Leu Gly Leu Ser Gln Leu Leu Gln Pro Glu GlyHis His Trp Glu Thr 130 135 140 Gln Gln Ile Pro Ser Leu Ser Pro Ser GlnPro Trp Gln Arg Leu Leu 145 150 155 160 Leu Arg Phe Lys Ile Leu Arg SerLeu Gln Ala Phe Val Ala Val Ala 165 170 175 Ala Arg Val Phe Ala His GlyAla Ala Thr Leu Ser Pro 180 185 <210> SEQ ID NO 2 <211> LENGTH: 1026<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (144)...(710) <400> SEQUENCE: 2 aactcggtgaacaactgagg gaaccaaacc agagacgcgc tgaacagaga gaatcaggct 60 caaagcaagtggaagtgggc agagattcca ccaggactgg tgcaaggcgc agagccagcc 120 agatttgagaagaaggcaaa aag atg ctg ggg agc aga gct gta atg ctg ctg 173 Met Leu GlySer Arg Ala Val Met Leu Leu 1 5 10 ttg ctg ctg ccc tgg aca gct cag ggcaga gct gtg cct ggg ggc agc 221 Leu Leu Leu Pro Trp Thr Ala Gln Gly ArgAla Val Pro Gly Gly Ser 15 20 25 agc cct gcc tgg act cag tgc cag cag ctttca cag aag ctc tgc aca 269 Ser Pro Ala Trp Thr Gln Cys Gln Gln Leu SerGln Lys Leu Cys Thr 30 35 40 ctg gcc tgg agt gca cat cca cta gtg gga cacatg gat cta aga gaa 317 Leu Ala Trp Ser Ala His Pro Leu Val Gly His MetAsp Leu Arg Glu 45 50 55 gag gga gat gaa gag act aca aat gat gtt ccc catatc cag tgt gga 365 Glu Gly Asp Glu Glu Thr Thr Asn Asp Val Pro His IleGln Cys Gly 60 65 70 gat ggc tgt gac ccc caa gga ctc agg gac aac agt cagttc tgc ttg 413 Asp Gly Cys Asp Pro Gln Gly Leu Arg Asp Asn Ser Gln PheCys Leu 75 80 85 90 caa agg atc cac cag ggt ctg att ttt tat gag aag ctgcta gga tcg 461 Gln Arg Ile His Gln Gly Leu Ile Phe Tyr Glu Lys Leu LeuGly Ser 95 100 105 gat att ttc aca ggg gag cct tct ctg ctc cct gat agccct gtg ggc 509 Asp Ile Phe Thr Gly Glu Pro Ser Leu Leu Pro Asp Ser ProVal Gly 110 115 120 cag ctt cat gcc tcc cta ctg ggc ctc agc caa ctc ctgcag cct gag 557 Gln Leu His Ala Ser Leu Leu Gly Leu Ser Gln Leu Leu GlnPro Glu 125 130 135 ggt cac cac tgg gag act cag cag att cca agc ctc agtccc agc cag 605 Gly His His Trp Glu Thr Gln Gln Ile Pro Ser Leu Ser ProSer Gln 140 145 150 cca tgg cag cgt ctc ctt ctc cgc ttc aaa atc ctt cgcagc ctc cag 653 Pro Trp Gln Arg Leu Leu Leu Arg Phe Lys Ile Leu Arg SerLeu Gln 155 160 165 170 gcc ttt gtg gct gta gcc gcc cgg gtc ttt gcc catgga gca gca acc 701 Ala Phe Val Ala Val Ala Ala Arg Val Phe Ala His GlyAla Ala Thr 175 180 185 ctg agt ccc taaaggcagc agctcaagga tggcactcagatctccatgg 750 Leu Ser Pro cccagcaagg ccaagataaa tctaccaccc caggcacctgtgagccaaca ggttaattag 810 tccattaatt ttagtgggac ctgcatatgt tgaaaattaccaatactgac tgacatgtga 870 tgctgaccta tgataaggtt gagtatttat tagatgggaagggaaatttg gggattattt 930 atcctcctgg ggacagtttg gggaggatta tttattgtatttatattgaa ttatgtactt 990 ttttcaataa agtcttattt ttgtggctaa aaaaaa 1026<210> SEQ ID NO 3 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Artificiallysynthesized primer sequence <400> SEQUENCE: 3 gagaagaggg agatgaagagactac 25 <210> SEQ ID NO 4 <211> LENGTH: 25 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Artificially synthesized primer sequence <400> SEQUENCE: 4 ctgagtccttgggggtcaca gccat 25 <210> SEQ ID NO 5 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 5gtgggacctg catatgttga aaatt 25 <210> SEQ ID NO 6 <211> LENGTH: 26 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 6ccccaaattt cccttcccat ctaata 26 <210> SEQ ID NO 7 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 7ccctactggg cctcagccaa ctcct 25 <210> SEQ ID NO 8 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 8ggagcagaga aggctcccct gtgaa 25 <210> SEQ ID NO 9 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 9gggcagagat tccaccagga ctggt 25 <210> SEQ ID NO 10 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 10ccagtcctgg tggaatctct gccca 25 <210> SEQ ID NO 11 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 11gaagctctgc acactggcct ggagt 25 <210> SEQ ID NO 12 <211> LENGTH: 25 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 12cactccaggc cagtgtgcag agctt 25 <210> SEQ ID NO 13 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 13ctgaagggct atggtggaga a 21 <210> SEQ ID NO 14 <211> LENGTH: 32 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 14ttgaattcca ccatgctggg gagcagagct gt 32 <210> SEQ ID NO 15 <211> LENGTH:28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Artificially synthesized primer sequence <400>SEQUENCE: 15 aaagatctta gggactcagg gttgctgc 28 <210> SEQ ID NO 16 <211>LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Artificially synthesized primersequence <400> SEQUENCE: 16 aagaattcta gggactcagg gttgctgc 28 <210> SEQID NO 17 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Artificiallysynthesized primer sequence <400> SEQUENCE: 17 ggtttaaata tttgttctcccttacccc 28 <210> SEQ ID NO 18 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Artificially synthesized primer sequence <400> SEQUENCE: 18 ttcagctgcttgggaggctg aggcagg 27 <210> SEQ ID NO 19 <211> LENGTH: 31 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Artificially synthesized primer sequence <400> SEQUENCE: 19aggaattcca ccaggactgg tgcaaggcgc a 31 <210> SEQ ID NO 20 <211> LENGTH:34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Artificially synthesized primer sequence <400>SEQUENCE: 20 gtctcgagaa aatatcattc tccaccatag ccct 34 <210> SEQ ID NO 21<211> LENGTH: 2398 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (461)...(622) <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (842)...(940) <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1107)...(1253) <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1359)...(1517) <220>FEATURE: <221> NAME/KEY: exon <222> LOCATION: (318)...(622) <220>FEATURE: <221> NAME/KEY: intron <222> LOCATION: (623)...(841) <220>FEATURE: <221> NAME/KEY: exon <222> LOCATION: (842)...(940) <220>FEATURE: <221> NAME/KEY: intron <222> LOCATION: (941)...(1106) <220>FEATURE: <221> NAME/KEY: exon <222> LOCATION: (1107)...(1253) <220>FEATURE: <221> NAME/KEY: intron <222> LOCATION: (1254)...(1358) <220>FEATURE: <221> NAME/KEY: exon <222> LOCATION: (1359)...(1826) <220>FEATURE: <221> NAME/KEY: TATA_signal <222> LOCATION: (238)...(242) <220>FEATURE: <221> NAME/KEY: polyA_signal <222> LOCATION: (1803)...(1807)<400> SEQUENCE: 21 tttaaatatt tgttctccct tacccctccc accccatccccgctgtgccc cccatccccg 60 ccccttctat agctatttcg attcctggag agcattacacatgtgtccca tcccaggcct 120 ctagccacag caaccacact actcatttcc cctggaactgaggctgcata cctgggctcc 180 ccacagaggg ggatgatgca gggaggggaa tcccacctgctgtgagtcac ctgctggtat 240 aaagggcggg ccttacaatg cagggacctt aaaagactcagagacaaagg gagaaaaaca 300 acaggaagca gcttacaaac tcggtgaaca actgagggaaccaaaccaga gacgcgctga 360 acagagagaa tcaggctcaa agcaagtgga agtgggcagagattccacca ggactggtgc 420 aaggcgcaga gccagccaga tttgagaaga aggcaaaaagatg ctg ggg agc aga 475 Met Leu Gly Ser Arg 1 5 gct gta atg ctg ctg ttgctg ctg ccc tgg aca gct cag ggc aga gct 523 Ala Val Met Leu Leu Leu LeuLeu Pro Trp Thr Ala Gln Gly Arg Ala 10 15 20 gtg cct ggg ggc agc agc cctgcc tgg act cag tgc cag cag ctt tca 571 Val Pro Gly Gly Ser Ser Pro AlaTrp Thr Gln Cys Gln Gln Leu Ser 25 30 35 cag aag ctc tgc aca ctg gcc tggagt gca cat cca cta gtg gga cac 619 Gln Lys Leu Cys Thr Leu Ala Trp SerAla His Pro Leu Val Gly His 40 45 50 atg gtgagtggca gcccctggagcctaacagga gtccaggctc tccaaggctg 672 Met tggcagaaga ccgtgaccttgagtggaagc tggagggttg aaggccatta gggagtaaga 732 gaggacaaga gagtagggttcctgggagag tcatgggcct gagggtccag gttggcttca 792 gaagtactat cttacttcttcattctttcc acctcttcct tcattccag gat cta aga 850 Asp Leu Arg 55 gaa gaggga gat gaa gag act aca aat gat gtt ccc cat atc cag tgt 898 Glu Glu GlyAsp Glu Glu Thr Thr Asn Asp Val Pro His Ile Gln Cys 60 65 70 gga gat ggctgt gac ccc caa gga ctc agg gac aac agt cag 940 Gly Asp Gly Cys Asp ProGln Gly Leu Arg Asp Asn Ser Gln 75 80 85 gtaccactgg gatgtggctgggcaatgaag gagaggggac tgagaacatg gctgggtacc 1000 atggtaaacc agaagttgtgtctgaaaata gtaagaaact gggtgagtct tcagtgaatg 1060 gagtaggaag agggtgtcctctttcattgc tttcttttct ccctag ttc tgc ttg 1115 Phe Cys Leu 90 caa agg atccac cag ggt ctg att ttt tat gag aag ctg cta gga tcg 1163 Gln Arg Ile HisGln Gly Leu Ile Phe Tyr Glu Lys Leu Leu Gly Ser 95 100 105 gat att ttcaca ggg gag cct tct ctg ctc cct gat agc cct gtg ggc 1211 Asp Ile Phe ThrGly Glu Pro Ser Leu Leu Pro Asp Ser Pro Val Gly 110 115 120 cag ctt catgcc tcc cta ctg ggc ctc agc caa ctc ctg cag 1253 Gln Leu His Ala Ser LeuLeu Gly Leu Ser Gln Leu Leu Gln 125 130 135 gtatgaagta ggggcgtggaggatgggggc ttgcaggtgt cagagacaga gggttggggg 1313 ttaagggttt agagtcttctctgactgtgt cctatgtcct ttcag cct gag ggt cac 1370 Pro Glu Gly His 140 cactgg gag act cag cag att cca agc ctc agt ccc agc cag cca tgg 1418 His TrpGlu Thr Gln Gln Ile Pro Ser Leu Ser Pro Ser Gln Pro Trp 145 150 155 cagcgt ctc ctt ctc cgc ttc aaa atc ctt cgc agc ctc cag gcc ttt 1466 Gln ArgLeu Leu Leu Arg Phe Lys Ile Leu Arg Ser Leu Gln Ala Phe 160 165 170 gtggct gta gcc gcc cgg gtc ttt gcc cat gga gca gca acc ctg agt 1514 Val AlaVal Ala Ala Arg Val Phe Ala His Gly Ala Ala Thr Leu Ser 175 180 185 ccctaaaggcagc agctcaagga tggcactcag atctccatgg cccagcaagg 1567 Proccaagataaa tctaccaccc caggcacctg tgagccaaca ggttaattag tccattaatt 1627ttagtgggac ctgcatatgt tgaaaattac caatactgac tgacatgtga tgctgaccta 1687tgataaggtt gagtatttat tagatgggaa gggaaatttg gggattattt atcctcctgg 1747ggacagtttg gggaggatta tttattgtat ttatattgaa ttatgtactt ttttcaataa 1807agtcttattt ttgtggctat atgagtctaa tttctaggct caattgggaa agagaaatcg 1867atggaaaaat aaggccaaga gactacaata tgcatccctt tcttctattc tgaagggcta 1927tggtggagaa tgatattttc tcatgacccc ctggtgtata gaataactgg gatctcttta 1987gtattaattc ctatatggct gagcaagcag aatgggatta ccagattagg aagtgggatc 2047atacctaagg gtcacttgct ccctgatcca gtgtctcctt ccctgctttc ttggccaaga 2107gtatatctga tcaaagacgg gagtcctgat cattgcagga tcaaaagtca gagttcagct 2167ttgagcagga agggcattcc agggaaatga agataaatat cctagaataa tgggactttc 2227ctctcaaagg acaattggaa tccctttttt tttttttttt tttttttttt tttttgagat 2287ggagtctcat tctgttgccc aggctggagt gcagtggcgt gatctctgct cactgcaacc 2347tccgcctccc acgttgaagc gattctcctg cctcagcctc ccaagcagct g 2398 <210> SEQID NO 22 <211> LENGTH: 54 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 22 Met Leu Gly Ser Arg Ala Val Met Leu Leu Leu Leu LeuPro Trp Thr 1 5 10 15 Ala Gln Gly Arg Ala Val Pro Gly Gly Ser Ser ProAla Trp Thr Gln 20 25 30 Cys Gln Gln Leu Ser Gln Lys Leu Cys Thr Leu AlaTrp Ser Ala His 35 40 45 Pro Leu Val Gly His Met 50 <210> SEQ ID NO 23<211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 23 Asp Leu Arg Glu Glu Gly Asp Glu Glu Thr Thr Asn Asp Val ProHis 1 5 10 15 Ile Gln Cys Gly Asp Gly Cys Asp Pro Gln Gly Leu Arg AspAsn Ser 20 25 30 Gln <210> SEQ ID NO 24 <211> LENGTH: 49 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 24 Phe Cys Leu Gln Arg IleHis Gln Gly Leu Ile Phe Tyr Glu Lys Leu 1 5 10 15 Leu Gly Ser Asp IlePhe Thr Gly Glu Pro Ser Leu Leu Pro Asp Ser 20 25 30 Pro Val Gly Gln LeuHis Ala Ser Leu Leu Gly Leu Ser Gln Leu Leu 35 40 45 Gln <210> SEQ ID NO25 <211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400>SEQUENCE: 25 Pro Glu Gly His His Trp Glu Thr Gln Gln Ile Pro Ser Leu SerPro 1 5 10 15 Ser Gln Pro Trp Gln Arg Leu Leu Leu Arg Phe Lys Ile LeuArg Ser 20 25 30 Leu Gln Ala Phe Val Ala Val Ala Ala Arg Val Phe Ala HisGly Ala 35 40 45 Ala Thr Leu Ser Pro 50 <210> SEQ ID NO 26 <211> LENGTH:26 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Consensus sequence <220> FEATURE: <221>NAME/KEY: VARIANT <222> LOCATION: 2-10, 12-17,20-21, 23-25 <223> OTHERINFORMATION: Xaa = any amino acid <220> FEATURE: <221> NAME/KEY: VARIANT<222> LOCATION: 22 <223> OTHER INFORMATION: Xaa = Phe, or Tyr <400>SEQUENCE: 26 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa XaaXaa 1 5 10 15 Xaa Gly Leu Xaa Xaa Xaa Xaa Xaa Xaa Leu 20 25 <210> SEQ IDNO 27 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (1) <223> OTHERINFORMATION: Xaa = unsure <400> SEQUENCE: 27 Xaa Ala Val Pro Gly Gly Ser1 5 <210> SEQ ID NO 28 <211> LENGTH: 55 <212> TYPE: PRT <213> ORGANISM:Mustela vison <400> SEQUENCE: 28 Ala Glu Asn Asn Leu Lys Leu Pro Lys LeuAla Glu Lys Asp Lys Cys 1 5 10 15 Phe Gln Ser Gln Phe Asn Gln Glu ThrCys Met Thr Arg Ile Thr Thr 20 25 30 Gly Leu Gln Glu Phe Gln Ile His LeuLys Tyr Leu Glu Ala Asn Tyr 35 40 45 Glu Gly Asn Lys Asn Asn Ala 50 55<210> SEQ ID NO 29 <211> LENGTH: 89 <212> TYPE: PRT <213> ORGANISM:Capra hircus <400> SEQUENCE: 29 Lys Thr Glu Ala Leu Ile Lys His Ile ValAsp Lys Ile Ser Ala Ile 1 5 10 15 Arg Lys Glu Ile Cys Glu Lys Asn AspGlu Cys Glu Asn Ser Lys Glu 20 25 30 Thr Leu Ala Glu Asn Lys Leu Lys LeuPro Lys Met Glu Glu Lys Asp 35 40 45 Gly Cys Phe Gln Ser Gly Phe Asn GlnAla Ile Cys Leu Ile Lys Thr 50 55 60 Thr Ala Gly Leu Leu Glu Tyr Gln IleTyr Leu Asp Phe Leu Gln Asn 65 70 75 80 Glu Phe Glu Gly Asn Gln Glu ThrVal 85 <210> SEQ ID NO 30 <211> LENGTH: 89 <212> TYPE: PRT <213>ORGANISM: Ovis aries <400> SEQUENCE: 30 Lys Thr Glu Ala Leu Ile Lys HisIle Val Asp Lys Ile Ser Ala Ile 1 5 10 15 Arg Lys Glu Ile Cys Glu LysAsn Asp Glu Cys Glu Asn Ser Lys Glu 20 25 30 Thr Leu Ala Glu Asn Lys LeuLys Leu Pro Lys Met Glu Glu Lys Asp 35 40 45 Gly Cys Phe Gln Ser Gly PheAsn Gln Ala Ile Cys Leu Ile Lys Thr 50 55 60 Thr Ala Gly Leu Leu Glu TyrGln Ile Tyr Leu Asp Phe Leu Gln Asn 65 70 75 80 Glu Phe Glu Gly Asn GlnGlu Thr Val 85 <210> SEQ ID NO 31 <211> LENGTH: 89 <212> TYPE: PRT <213>ORGANISM: Bos taurus <400> SEQUENCE: 31 Lys Thr Glu Ala Leu Ile Lys ArgMet Val Asp Lys Ile Ser Ala Met 1 5 10 15 Arg Lys Glu Ile Cys Glu LysAsn Asp Glu Cys Glu Ser Ser Lys Glu 20 25 30 Thr Leu Ala Glu Asn Lys LeuAsn Leu Pro Lys Met Glu Glu Lys Asp 35 40 45 Gly Cys Phe Gln Ser Gly PheAsn Gln Ala Ile Cys Leu Ile Arg Thr 50 55 60 Thr Ala Gly Leu Leu Glu TyrGln Ile Tyr Leu Asp Tyr Leu Gln Asn 65 70 75 80 Glu Tyr Glu Gly Asn GlnGlu Asn Val 85 <210> SEQ ID NO 32 <211> LENGTH: 89 <212> TYPE: PRT <213>ORGANISM: Equus caballus <400> SEQUENCE: 32 Lys Thr Lys Gln His Ile LysTyr Ile Leu Gly Lys Ile Ser Ala Leu 1 5 10 15 Lys Asn Glu Met Cys AsnAsn Phe Ser Lys Cys Glu Asn Ser Lys Glu 20 25 30 Val Leu Ala Glu Asn AsnLeu Asn Leu Pro Lys Met Ala Glu Lys Asp 35 40 45 Gly Cys Phe Gln Ser GlyPhe Asn Gln Glu Thr Cys Leu Met Lys Ile 50 55 60 Thr Thr Gly Leu Ser GluPhe Gln Ile Tyr Leu Glu Tyr Leu Gln Asn 65 70 75 80 Glu Phe Lys Gly GluLys Glu Asn Ile 85 <210> SEQ ID NO 33 <211> LENGTH: 89 <212> TYPE: PRT<213> ORGANISM: Sus scrofa <400> SEQUENCE: 33 Lys Thr Glu Glu Leu IleLys Tyr Ile Leu Gly Lys Ile Ser Ala Met 1 5 10 15 Arg Lys Glu Met CysGlu Lys Tyr Glu Lys Cys Glu Asn Ser Lys Glu 20 25 30 Val Leu Ala Glu AsnAsn Leu Asn Leu Pro Lys Met Ala Glu Lys Asp 35 40 45 Gly Cys Phe Gln SerGly Phe Asn Gln Glu Thr Cys Leu Met Arg Ile 50 55 60 Thr Thr Gly Leu ValGlu Phe Gln Ile Tyr Leu Asp Tyr Leu Gln Lys 65 70 75 80 Glu Tyr Glu SerAsn Lys Gly Asn Val 85 <210> SEQ ID NO 34 <211> LENGTH: 89 <212> TYPE:PRT <213> ORGANISM: Canis familiaris <400> SEQUENCE: 34 Lys Val Glu GluLeu Ile Lys Tyr Ile Leu Gly Lys Ile Ser Ala Leu 1 5 10 15 Arg Lys GluMet Cys Asp Lys Phe Asn Lys Cys Glu Asp Ser Lys Glu 20 25 30 Ala Leu AlaGlu Asn Asn Leu His Leu Pro Lys Leu Glu Gly Lys Asp 35 40 45 Gly Cys PheGln Ser Gly Phe Asn Gln Glu Thr Cys Leu Thr Arg Ile 50 55 60 Thr Thr GlyLeu Val Glu Phe Gln Leu His Leu Asn Ile Leu Gln Asn 65 70 75 80 Asn TyrGlu Gly Asp Lys Glu Asn Val 85 <210> SEQ ID NO 35 <211> LENGTH: 89 <212>TYPE: PRT <213> ORGANISM: Felis catus <400> SEQUENCE: 35 Lys Met Glu GluLeu Ile Lys Tyr Ile Leu Gly Lys Ile Ser Ala Leu 1 5 10 15 Lys Lys GluMet Cys Asp Asn Tyr Asn Lys Cys Glu Asp Ser Lys Glu 20 25 30 Ala Leu AlaGlu Asn Asn Leu Asn Leu Pro Lys Leu Ala Glu Lys Asp 35 40 45 Gly Cys PheGln Ser Gly Phe Asn Gln Glu Thr Cys Leu Thr Arg Ile 50 55 60 Thr Thr GlyLeu Gln Glu Phe Gln Ile Tyr Leu Lys Phe Leu Gln Asp 65 70 75 80 Lys TyrGlu Gly Asp Glu Glu Asn Ala 85 <210> SEQ ID NO 36 <211> LENGTH: 89 <212>TYPE: PRT <213> ORGANISM: Cercocebus torquatus atys <400> SEQUENCE: 36Arg Ile Asp Lys His Ile Arg Tyr Ile Leu Asp Gly Ile Ser Ala Leu 1 5 1015 Arg Lys Glu Thr Cys Asn Arg Ser Asn Met Cys Asp Ser Thr Lys Glu 20 2530 Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala Glu Lys Asp 35 4045 Gly Cys Phe Gln Ser Gly Phe Asn Glu Asp Thr Cys Leu Val Lys Ile 50 5560 Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr Leu Gln Asn 65 7075 80 Arg Phe Glu Ser Ser Glu Glu Gln Ala 85 <210> SEQ ID NO 37 <211>LENGTH: 89 <212> TYPE: PRT <213> ORGANISM: Macaca mulatta <400>SEQUENCE: 37 Arg Ile Asp Lys His Ile Arg Tyr Ile Leu Asp Gly Ile Ser AlaLeu 1 5 10 15 Arg Lys Glu Thr Cys Asn Arg Ser Asn Met Cys Glu Ser SerLys Glu 20 25 30 Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys Met Ala GluLys Asp 35 40 45 Gly Cys Phe Gln Ser Gly Phe Asn Glu Asp Thr Cys Leu ValLys Ile 50 55 60 Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr LeuGln Asn 65 70 75 80 Arg Phe Glu Ser Ser Glu Glu Gln Ala 85 <210> SEQ IDNO 38 <211> LENGTH: 89 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 38 Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly IleSer Ala Leu 1 5 10 15 Arg Lys Glu Thr Cys Asn Lys Ser Asn Met Cys GluSer Ser Lys Glu 20 25 30 Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro Lys MetAla Glu Lys Asp 35 40 45 Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu Thr CysLeu Val Lys Ile 50 55 60 Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu GluTyr Leu Gln Asn 65 70 75 80 Arg Phe Glu Ser Ser Glu Glu Gln Ala 85 <210>SEQ ID NO 39 <211> LENGTH: 89 <212> TYPE: PRT <213> ORGANISM: Musmusculus <400> SEQUENCE: 39 Gln Val Gly Gly Leu Ile Thr His Val Leu TrpGlu Ile Val Glu Met 1 5 10 15 Arg Lys Glu Leu Cys Asn Gly Asn Ser AspCys Met Asn Asn Asp Asp 20 25 30 Ala Leu Ala Glu Asn Asn Leu Lys Leu ProGlu Ile Gln Arg Asn Asp 35 40 45 Gly Cys Tyr Gln Thr Gly Tyr Asn Gln GluIle Cys Leu Leu Lys Ile 50 55 60 Ser Ser Gly Leu Leu Glu Tyr His Ser TyrLeu Glu Tyr Met Lys Asn 65 70 75 80 Asn Leu Lys Asp Asn Lys Lys Asp Lys85 <210> SEQ ID NO 40 <211> LENGTH: 89 <212> TYPE: PRT <213> ORGANISM:Rattus norvegicus <400> SEQUENCE: 40 Gln Val Gly Gly Leu Ile Thr Tyr ValLeu Arg Glu Ile Leu Glu Met 1 5 10 15 Arg Lys Glu Leu Cys Asn Gly AsnSer Asp Cys Met Asn Ser Asp Asp 20 25 30 Ala Leu Ser Glu Asn Asn Leu LysLeu Pro Glu Ile Gln Arg Asn Asp 35 40 45 Gly Cys Phe Gln Thr Gly Tyr AsnGln Glu Ile Cys Leu Leu Lys Ile 50 55 60 Cys Ser Gly Leu Leu Glu Phe ArgPhe Tyr Leu Glu Phe Val Lys Asn 65 70 75 80 Asn Leu Gln Asp Asn Lys LysAsp Lys 85 <210> SEQ ID NO 41 <211> LENGTH: 88 <212> TYPE: PRT <213>ORGANISM: Bos taurus <400> SEQUENCE: 41 Lys Cys Leu Glu Gln Val Arg LysIle Gln Ala Asp Gly Ala Glu Leu 1 5 10 15 Gln Glu Arg Leu Cys Ala AlaHis Lys Leu Cys His Pro Glu Glu Leu 20 25 30 Met Leu Leu Arg His Ser LeuGly Ile Pro Gln Ala Pro Leu Ser Ser 35 40 45 Cys Ser Ser Gln Ser Leu GlnLeu Arg Gly Cys Leu Asn Gln Leu His 50 55 60 Gly Gly Leu Phe Leu Tyr GlnGly Leu Leu Gln Ala Leu Ala Gly Ile 65 70 75 80 Ser Pro Glu Leu Ala ProThr Le 85 <210> SEQ ID NO 42 <211> LENGTH: 88 <212> TYPE: PRT <213>ORGANISM: Canis familiaris <400> SEQUENCE: 42 Lys Cys Leu Glu Gln MetArg Lys Val Gln Ala Asp Gly Thr Ala Leu 1 5 10 15 Gln Glu Thr Leu CysAla Thr His Gln Leu Cys His Pro Glu Glu Leu 20 25 30 Val Leu Leu Gly HisAla Leu Gly Ile Pro Gln Pro Pro Leu Ser Ser 35 40 45 Cys Ser Ser Gln AlaLeu Gln Leu Met Gly Cys Leu Arg Gln Leu His 50 55 60 Ser Gly Leu Phe LeuTyr Gln Gly Leu Leu Gln Ala Leu Ala Gly Ile 65 70 75 80 Ser Pro Glu LeuAla Pro Thr Leu 85 <210> SEQ ID NO 43 <211> LENGTH: 88 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 43 Lys Cys Leu Glu Gln ValArg Lys Ile Gln Gly Asp Gly Ala Ala Leu 1 5 10 15 Gln Glu Lys Leu CysAla Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 20 25 30 Val Leu Leu Gly HisSer Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 35 40 45 Cys Pro Ser Gln AlaLeu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 50 55 60 Ser Gly Leu Phe LeuTyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 65 70 75 80 Ser Pro Glu LeuGly Pro Thr Leu 85 <210> SEQ ID NO 44 <211> LENGTH: 88 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 44 Lys Ser Leu Glu Gln ValArg Lys Ile Gln Ala Ser Gly Ser Val Leu 1 5 10 15 Leu Glu Gln Leu CysAla Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 20 25 30 Val Leu Leu Gly HisSer Leu Gly Ile Pro Lys Ala Ser Leu Ser Gly 35 40 45 Cys Ser Ser Gln AlaLeu Gln Gln Thr Gln Cys Leu Ser Gln Leu His 50 55 60 Ser Gly Leu Cys LeuTyr Gln Gly Leu Leu Gln Ala Leu Ser Gly Ile 65 70 75 80 Ser Pro Ala LeuAla Pro Thr Leu 85 <210> SEQ ID NO 45 <211> LENGTH: 88 <212> TYPE: PRT<213> ORGANISM: Gallus gallus <400> SEQUENCE: 45 Lys Asn Leu Glu Phe ThrArg Lys Ile Arg Gly Asp Val Ala Ala Leu 1 5 10 15 Gln Arg Ala Val CysAsp Thr Phe Gln Leu Cys Thr Glu Glu Glu Leu 20 25 30 Gln Leu Val Gln ProAsp Pro His Leu Val Gln Ala Pro Leu Asp Gln 35 40 45 Cys His Lys Arg GlyPhe Gln Ala Glu Val Cys Phe Thr Gln Ile Arg 50 55 60 Ala Gly Leu His AlaTyr His Asp Ser Leu Gly Ala Val Leu Arg Leu 65 70 75 80 Leu Pro Asn HisThr Thr Leu Val 85 <210> SEQ ID NO 46 <211> LENGTH: 89 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Consensus sequence <400> SEQUENCE: 46 Lys Cys Leu Glu MetIle Arg Tyr Ile Leu Gly Asp Ile Ser Ala Leu 1 5 10 15 Arg Lys Glu LeuCys Asp Thr Tyr Gln Leu Cys His Asn Glu Glu Glu 20 25 30 Val Leu Ala GluAsn Asn Leu Asn Leu Pro Lys Met Ala Glu Lys Asp 35 40 45 Gly Cys Phe GlnSer Gly Phe Asn Gln Glu Thr Cys Leu Thr Gln Ile 50 55 60 Thr Thr Gly LeuMet Glu Tyr Gln Ile Tyr Leu Glu Tyr Leu Gln Asn 65 70 75 80 Asn Tyr ProGly Asn Lys Glu Asn Val 85

What is claimed is
 1. A substantially pure polypeptide comprising anamino acid sequence at least 400/) identical to SEQ ID NO:1, wherein thepolypeptide promotes proliferation of a cell that is Lin negative, Sca-1positive, and c-kit positive.
 2. The polypeptide of claim 1, wherein theamino acid sequence is at least 60% identical to SEQ ID NO:1.
 3. Thepolypeptide of claim 1, wherein the amino acid sequence is at least 80%identical to SEQ ID NO:1.
 4. The polypeptide of claim 1, wherein theamino acid sequence is at least 90% identical to SEQ ID NO:1.
 5. Asubstantially pure polypeptide comprising the sequence of SEQ ID NO:1.6. A substantially pure polypeptide comprising the amino acid sequenceof SEQ ID NO:1, with up to 30 conservative amino acid substitutions,wherein the polypeptide promotes proliferation of a cell that is Linnegative, Sca-1 positive, and c-kit positive.
 7. A substantially purepolypeptide encoded by a nucleic acid that hybridizes under-highstringency conditions to a probe the sequence of which consists of SEQID NO:2, wherein the polypeptide promotes proliferation of a cell thatis Lin negative, Sca-1 positive, and c-kit positive.
 8. An isolatednucleic acid encoding the polypeptide of claim
 1. 9. An isolated nucleicacid encoding the polypeptide of claim
 5. 10. An isolated nucleic acidencoding the polypeptide of claim
 6. 11. An isolated nucleic acidcomprising a strand that hybridizes under high stringency conditions toa single stranded probe, the sequence of which consists of SEQ ID NO:2or the complement of SEQ ID NO:2.
 12. The isolated nucleic acid of claim11, wherein the nucleic acid enicocdes a polypeptide that promotesproliferation of a cell that is Lin negative, Sca-1 positive, and c-kitpositive.
 13. The nucleic acid of claim 12, wherein the amino acidsequence of the polypeptide comprises SEQ ID NO:1.
 14. The nucleic acidof claim 11, wherein the strand is at least 15 nucleotides in length.15. The nucleic acid of claim 14, wherein the nucleic acid is anantisense nucleic acid that inhibits expression of a polypeptidecomprising SEQ ID NO:1.
 16. The nucleic acid of claim 15, wherein thenucleic acid is at least 15 nucleotides in length.
 17. A vectorcomprising the nucleic acid of claim
 8. 18. A vector comprising thenucleic acid of claim
 9. 19. A vector comprising the nucleic acid ofclaim
 10. 20. A vector comprising the nucleic acid of claim
 11. 21. Avector comprising the nucleic acid of claim
 12. 22. A cultured host cellcomprising the nucleic acid of claim
 8. 23. A cultured host cellcomprising the nucleic acid of claim
 9. 24. A cultured host cellcomprising the nucleic acid of claim
 10. 25. A cultured host cellcomprising the nucleic acid of claiml
 11. 26. A cultured host cellcomprising the nucleic acid of claim
 12. 27. A method of producing apolypeptide, the method comprising culturing the cultured host cell ofclaim 22 in a culture, expressing the polypeptide in the cultured hostcell, and isolating the polypeptide from the culture.
 28. A method ofscreening for a compound that binds to the polypeptide of claim 1, themethod comprising: contacting a test sample with the polypeptide or apartial peptide thereof, detecting the binding activity of the testsample to the polypeptide or a partial peptide thereof, and selecting acompound binding to the polypeptide or a partial peptide thereof
 29. Acompound which specifically binds to the polypeptide of claim
 1. 30. Thecompound of claim 29, wherein the compound is obtained by the method ofclaim
 28. 31. A method for screening a compound that promotes orinhibits an activity of the polypeptide of claim 1, the methodcomprising contacting a mammalian bone marrow cell with the polypeptideof claim 1 and a kit ligand, in the presence of a test compound;detecting the proliferation of the bone marrow cell; and selecting acompound that promotes or inhibits the proliferation of a bone marrowcell in comparison with a bone marrow cell cultured in the presence ofthe test compound.
 32. The method of claim 31, wherein the bone marrowcell is Lin negative, Sca-1 positive, c-kit positive, and CD34 positive.33. A compound that promotes or inhibits an activity of the polypeptideof claim
 1. 34. The compound of claim 33, wherein the compound isobtained by the method of claim
 31. 35. A pharmaceutical compositioncomprising the polypeptide of claim 1 as an active ingredient.
 36. Apromoter or inhibitor of the polypeptide of claim 1, wherein the activecomponent is the compound of claim
 33. 37. An antibody that specificallybinds to the polypeptide of claim 1.